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Supporting information
An Affinity-based Probe for Methyltransferase Enzymes Based on Sinefungin
Matthew A. Lafreniere1, Genevieve F. Desrochers1, Kedous Mekbib1, and John Paul Pezacki1,2*
1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie
Street, Ottawa, ON, Canada, K1N 6N5
2. Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451
Smyth Road, Ottawa, ON, Canada, K1H 8M5
* Corresponding author: [email protected]
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Table
Experimental methods and procedures 3-7
Supporting figures and tables 8-10
Synthesis and spectral data 11-20
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Tissue culture and preparation of cell lysates
Hek293 cells were grown and maintained in MEM medium (GIBCO-BRL, Burlington, Ontario)
and supplemented with 50ml of 10% fetal bovine serum (FBS, PAA Laboratories), 5ml of
50U/ml penicillin/streptomycin, and 5ml of Sodium Pyruvate (GIBCO). For active proteome
extraction, subconfluent Hek293 cells were washed twice with phosphate buffered saline (PBS) ,
followed by the addition of1ml of 0.05% Triton-X in PB. The cell lysates were scrapped, pooled,
and kept at 0oC. Lysates were sonicated (15 pulses/sample, 50 % duty cycle; Sonifier 250,
Branson Ultrasonic, Danbury, CT) and centrifuged for 15 min (14,000 rpm, 4oC). The
supernatant was transferred to a sterile Eppendorf tube and quantified via DC protein assay
(BioRad).
In vitro protein labeling
For in vitro protein labeling, various concentrations of BpyneSF were added to fresh cell lysates
in 100 μL 0.05 % Triton-X in PBS. Lysate samples were incubated with the probe for 5 minutes,
followed by UV irradiation at 365nm for 5 minutes on ice. After the UV irradiation, 100 μL of
freshly prepared click chemistry mix in PBS consisting of Rhodamine-azide (100 μM, 100 mM
stock solution in DMSO), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA, 200 μM,
50 mM stock solution in 4:1 DMSO:tert-butanol), copper sulfate (2 mM, 50 mM freshly
prepared stock solution in deionized water), and tris(2-carboxyethyl)phosphine hydrochloride
(TCEP, 2 mM, 50 mM freshly prepared stock in deionized water) were added and incubated for
15 minutes at room temperature with gentle mixing. The addition of 1000 μL of acetone
terminated the reaction and the samples were stored in a -80 ⁰C for at least 30 minutes. After at
least 30 minutes, protein precipitation was done by centrifugation for 15 min at 4 ⁰C and 14 000
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rpm. Acetone was removed and the protein pellets were allowed to dry for 5 minutes followed by
the addition of 35 μL of 2x Laemmli buffer containing beta-mercaptoethanol. Samples were
shaken for 15 minutes and heated for 5 minutes at 95oC. The samples were separated by 10%
SDS-PAGE (Bio-Rad, TGX Stain-FreeTM FastCastTM Acylamide Kit, 10%) and then visualized
by in-gel fluorescent scanning using ChemiDoc MP (Bio-Rad) imager. Gel visualization was
undertaken using both Coomassie blue staining and stain-free imaging, as previously described1.
Enrichment and mass spectrometry of labelled proteins
Biotin-azide click chemistry, streptavidin-ennrichment, and trypsin digest of probe-labelled
proteomes was performed as previously described2. However, acetone precipitation step was
included following the incubation with click mix. Following protein enrichment, agarose-
streptavidin beads (Pierce) were washed three times with 100 μL PBS in a Biospin column.
Using 200 μL of PBS, beads were transferred to probe-labelling cell lysates and incubated for 90
min. For the on-bead digestion, the beads were washed five times with 50 mM ammonium
bicarbonate, transferred to an Eppendorf tube and were heated for 15 min at 65 ⁰C in 500 μL of
10 mM DTT in 50 mM ammonium bicarbonate (ABC). After 15 minutes, 25 μL of 500 mM
iodoacetamide was added and lysates were rotated in the dark for 30 min. Samples were
centrifuged for 2 min at 1400 rpm and 100 μL of ABC was added followed by 2 μL of 0.5
mg/mL of Trypsin was added. Samples were rotated at 37 ⁰C overnight, followed by bead
pelleting and the transfer of the supernatant for MS analysis. HPLC-ESI-MS/MS was used for all
MS analyses. The system consisted of an Agilent 1100 micro-HPLC system (Agilent
Technologies, Santa Clara, CA, USA) coupled with an LTQ-Orbitrap mass spectrometer
(ThermoFisher Scientific, San Jose, CA) equipped with a nano-electrospray interface operated in
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positive ion mode. Pre-column was packed in-house with reverse phase Magic C18AQ resins (5
μm; 120-Å pore size; Dr. Maisch GmbH, Ammerbuch, Germany), and analytical column of 75
μm × 100 mm was packed with reverse phase Magic C18AQ resins (1.9 μm; 120-Å pore size;
Dr. Maisch GmbH, Ammerbuch, Germany). The mobile phases consisted of 0.1 % (v/v) FA in
water as buffer A and 0.1 % (v/v) FA in acetonitrile as buffer B. The 4 ul sample was loaded
onto the pre-column using a buffer containing 98% A at a flow rate of 4 µL/min for 5 min.
Subsequently, a gradient from 5 % to 35 % buffer B was performed, at a flow rate of ~300
nL/min obtained from splitting a 20 µL/min through a restrictor, in 60 min. The MS method
consisted of one full MS scan from 350 to 1700 m/z followed by data-dependent MS/MS scan of
the 5 most intense ions, with dynamic exclusion repeat count of 2, and repeat duration of 90 s.
As well, for the experiments on the Orbitrap MS the full MS was in performed in the Orbitrap
analyzer with R = 60,000 defined at m/z 400, while the MS/MS analysis were performed in the
LTQ. To improve the mass accuracy, all the measurements in Orbitrap mass analyzer were
performed with internal recalibration (“Lock Mass”). On the Orbitrap, the charge state rejection
function was enabled, with single and “unassigned” charge states rejected.
Analysis of mass spectrometry data
Raw files where generated using LTQ-Orbitrap and processed and analyzed using MaxQuant,
Version 1.3.0.5 using the Uniprot fasta protein database (2013, July version). A maximum of two
missing trypsin cleavages were permitted. Precursor ion mass tolerances were set to 7 ppm, and
the fragment ion mass tolerance was 0.8 Da for the MS/MS spectra. If the identified peptide
sequences of one protein were contained within or equal to another protein’s peptide set, the
proteins were grouped together and reported as a single protein group. The false discovery rate
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(FDR) for peptide and protein was set to 1 % and a minimum length of six amino acids was used
for peptide identification. Normalized LFQ intensity was used for protein quantification.
In vitro recombinant protein labeling
1 µg of CARM1 (Sigma-Aldrich), SETD2 (Sigma-Aldrich), and PRMT1 (Sigma-Aldrich) were
dissolved in 100 μL 0.05 % Triton-X in PBS. Recombinant proteins were incubated with the
probe for 30 minutes, followed by UV irradiation at 365nm for 45 minutes on ice. After the UV
irradiation, 100 μL of freshly prepared click chemistry mix in PBS consisting of Rhodamine-
azide (100 μM, 100 mM stock solution in DMSO), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]
amine (TBTA, 200 μM, 50 mM stock solution in DMSO), copper sulfate (2 mM, 50 mM freshly
prepared stock solution in deionized water), and tris(2-carboxyethyl)phosphine hydrochloride
(TCEP, 2 mM, 50 mM freshly prepared stock in deionized water) were added and incubated for
45 minutes at room temperature with gentle mixing. The addition of 1000 μL of acetone
terminated the reaction and the samples were stored in a -80 ⁰C for at least 30 minutes. After at
least 30 minutes, protein were pelleted by centrifugation for 15 min at 4 ⁰C and 14 000 rpm.
Acetone was removed and the protein pellets were allowed to dry for 5 minutes then solubilised
in SDS-PAGE loading buffer (0.1 M Tris, pH 6.8, 10% glycerol, 4% SDS, 0.02%
bromophenol blue, 30 mM DTT). Samples were vortexed and heated for 5 minutes at 95oC.
The samples were separated by 10% SDS-PAGE (Bio-Rad, TGX Stain-FreeTM FastCastTM
Acylamide Kit, 10%) and then visualized by in-gel fluorescent scanning using ChemiDoc MP
(Bio-Rad) imager. Total protein visualization was undertaken using both Coomassie blue
staining and stain-free imaging, as previously described1.
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High-Performance Liquid Chromatography Mass Spectrometry
Thermo Easy nLC II coupled with an Orbitrap Q Exactive Plus mass spectrometer (Thermo
Scientific, San Jose, Ca) was used for HPLC-MS analysis of small molecules, using an Acclaim
PepMap RSLC 75 µm ID x 150 mm length separation column (Thermo Scientific, San Jose, Ca).
18 µL of the BpyneSF were injected and separated by the following gradient (A – 0.1 % formic
acid in H2O, B – 80 % acetonitrile, 0.1 % formic acid in H20) with the flow of 200 nl/min: 0.0-
80.0 min 0-40% B, 80.0-80.1 min 40-80% B, 80.1-90.0 min 80% B, 90.0-90.1 min 80-2% B, 90.1-
115.0 min 2% B. The following parameters were used: Nano-ESI conditions: spray voltage in
positive mode – 2000 V; ion transfer tube temperature – 275°C; S-lens RF level – 60. Initial scans
of small molecule precursors from 300 to 1500 m/z were performed at 60K resolution (at 200 m/z)
with a 2 × 105 ion count target and maximum injection time of 50 ms. Tandem MS was performed
by isolation at 0.7 Th with the quadrupole, HCD fragmentation with collision energy of 30% with
5% step, and normal scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and
the max injection time was 35 ms. Precursors with charge state 2–6 were sampled for MS2. The
dynamic exclusion duration was set to 60 s with a 10 ppm tolerance around the selected precursor
and its isotopes. The instrument was run in top speed mode with 4 seconds per cycles.
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Figure S-1. Imaging total protein content following in-gel fluorescence labeling with BpyneSF
using the gel-free imaging protocol (left) on the ChemiDoc MP imager (Bio-Rad) or coomassie
stain (right).
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Figure S-2. Imaging total protein content following in-gel fluorescence labeling with BpyneSF
using the gel-free imaging protocol on the ChemiDoc MP imager (Bio-Rad).
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Table 1. Identification of proteins labelled by BpyneSF using nLC-MS/MS methods
described.
Accession Description
H7BZ93 Histone-lysine N-methyltransferase SETD2 (Fragment) OS=Homo sapiens GN=SETD2 PE=1 SV=1 - [H7BZ93_HUMAN]
A0A087WWT3 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1 - [A0A087WWT3_HUMAN]
Q96L17 FMN2 protein (Fragment) OS=Homo sapiens GN=FMN2 PE=2 SV=2 - [Q96L17_HUMAN]
Q8NDY3 [Protein ADP-ribosylarginine] hydrolase-like protein 1 OS=Homo sapiens GN=ADPRHL1 PE=2 SV=1 - [ARHL1_HUMAN]
E2QRD4 Protein MMS22-like OS=Homo sapiens GN=MMS22L PE=1 SV=1 - [E2QRD4_HUMAN]
O75132 Zinc finger BED domain-containing protein 4 OS=Homo sapiens GN=ZBED4 PE=1 SV=2 - [ZBED4_HUMAN]
H7BZ93 Histone-lysine N-methyltransferase SETD2 (Fragment) OS=Homo sapiens GN=SETD2 PE=1 SV=1 - [H7BZ93_HUMAN]
A2IB45 Tudor domain containing 6 OS=Homo sapiens GN=TDRD6 PE=2 SV=1 - [A2IB45_HUMAN]
E9PS68 Pyruvate carboxylase, mitochondrial OS=Homo sapiens GN=PC PE=1 SV=1 - [E9PS68_HUMAN]
H7C1I9 Microtubule-associated serine/threonine-protein kinase 4 (Fragment) OS=Homo sapiens GN=MAST4 PE=1 SV=1 - [H7C1I9_HUMAN]
B4DZA1 cDNA FLJ60986, moderately similar to Homo sapiens golgi autoantigen, golgin subfamily a, 8A (GOLGA8A), transcript variant 3, mRNA OS=Homo sapiens PE=2 SV=1 - [B4DZA1_HUMAN]
Q4G1H2 Abnormal spindle-like microcephaly associated splice variant 3 OS=Homo sapiens GN=ASPM PE=2 SV=1 - [Q4G1H2_HUMAN]
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Synthesis
Reagents
All reagents and solvents were purchased from Sigma-Aldrich, unless otherwise noted, and used
without further purification. Deuterated solvents were purchased from Cambridge Isotope
Laboratories and thin layer chromatography was done using Analtech Uniplate ® silica gel plates
(60A F254, layer thickness 250µm). Flash column chromatography was performed by silica gel
(60A, particle size 40 to 63 μm). 1H NMR and 13C NMR spectra were recorded using a Bruker-
DRX-400 spectrometer at a frequency of 400.13 MHz for 1 H and 100.61 MHz for 13C and
processed with Bruker TOPSPIN 2.1 software. Chemical shifts are reported in parts per million
(δ) using residual solvent resonance as an internal reference. The following abbreviations were
used to designate chemical shift multiplicies: s = singlet, d = doublet, t = triplet, m = multiplet or
unresolved, br = broad single and J = coupling constant in Hz.
N-(4-(4-aminobenzyol)phenyl)hex-5-ynamide (Bpyne) was synthesized per previously
published reports3.
(BOC)2Sinefungin was synthesized per previously published reports4
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Bpyne-gly-BOC. In a 100 mL flask was added N-(tert-Butoxycarbonyl)glycine (Boc-gly-OH)
(743 mg, 4.31 mmol), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-
oxid hexafluorophosphate (HATU) (1.70 g, 4.47 mmol), triethylamine (1.15 mL, 8.07 mmol)
and DMF (40 mL) The mixture was stirred for 5 minutes and then Bpyne (866.3 mg, 2.83 mmol)
was added in one portion, and the reaction was stirred at 80oC under argon overnight. After
stirring overnight, the mixture was concentrated in vacuo and the residue was purified over silica
(60% ethyl acetate in hexane) to yield a yellow, crystalline solid (138.2 mg, 11%). 1H NMR (400
MHz, CDCl3) δ 9.07 (s, 1H), 8.41 (s, 1H), 7.64 (m, 8H), 5.75 (s, 1H), 4.00 (d, J = 5.8 Hz, 2H),
2.55 (t, J = 7.3 Hz, 2H), 2.27 (td, J = 6.7, 2.5 Hz, 2H), 1.97 (t, J = 2.5 Hz, 2H), 1.93 (q, J = 7.1
Hz, 1H), 1.44 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 194.95, 194.87, 171.61, 168.76, 149.95,
142.13, 141.62, 133.59, 141.62, 133.59, 133.32, 133.00, 131.46, 131.43, 120.80, 119.20, 119.12,
83.47, 69.65, 36.11, 28.42, 23.97, 17.95. LRMS m/z calcd for C26H29N3O5 (M+H): 464.21.
Found: 464.2.
Bpyne-gly-NH2. In a 5 mL flask was added Bpyne-gly-BOC (50 mg, 0.107 mmol) in CHCl3 (2
mL), followed by the addition of trifluoroacetic acid (TFA) (200 µL, 2.612 mmol). The reaction
was stirred for 3 h and then concentration in vacuo to yield a yellow, crystalline solid (31.5 mg,
80%). 1H NMR (400 MHz, CDCl3) δ 7.50 (m, 8H), 3.56 (s, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.06
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(td, J = 6.8, 2.6 Hz, 2H), 1.84 (t, J = 2.5 Hz, 2H), 1.69 (q, J = 7.1 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 196.07, 173.14, 143.33, 142.19, 133.81, 132.81, 132.93, 131.77, 131.74, 119.39, 83.64,
78.19, 77.87, 77.55, 69.60, 49.86, 36.07, 24.54, 18.21. LRMS m/z calcd for C21H21N3O3 (M+H):
364.158. Found: 364.2.
BpyneSF(BOC)2. In a 5 mL flask was added (BOC)2Sinefungin (29.9 mg, 0.051 mmol), 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU) (22.2 mg, 0.058 mmol), and DMF (1 mL). Then, diisopropylethylamine (DIPEA) (10
µL, 0.058) was added and the reaction was stirred for 20 minutes. Then, Bpyne-gly-NH2 (17.7
mg, 0.048 mmol) was added to the reaction mixture and the reaction was stirred overnight at
60oC under argon. After 24 h, product synthesis was confirmed through LC/MS (eluting 10 to 60
% acetonitrile/water with 0.1 % formic acid) and solution was concentration in vacuo. Residue
was purified on HPLC eluting 10 to 60 % acetonitrile/water with 0.1 % formic acid and yielded a
light yellow, crystalline solid (15.2 mg, 33.7%). 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H),
8.20 (s, 1H), 7.73 (m, 8H), 5.95 (d, J = 4.9 Hz, 1H), 4.83-4.76 (m, 2H), 4.16 (s, 2H), 4.05 (s,
2H), 3.70 (m, 1H), 3.35 (s, 1H) 2.56 (t, J =7.4 Hz, 2H), 2.28 (m, 3H), 2.02 (m, 1H), 1.90 (m,
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4H), 1.70 (m, 1H), 1.59 (m, 2H), 1.42 (s, 9H), 1.40 (s, 9H). LRMS m/z calcd for C46H58N10O11
(M+H): 927.43. Found: 927.8.
BpyneSF. In a 5 mL flask was added BpyneSF(BOC)2 (17.7 mg, 0.0019 mmol) and TFA (1
mL). The solution was stirred for 3 h and then the reaction mixture was concentrated in vacuo to
quantitatively yield a yellow, crystalline solid (13.8 mg). Synthesis was confirmed using
HPLC/MS. HRMS m/z calcd for C36H42N10O7 (M+H): 727.32. Found: 727.33269.
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